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Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production

Abstract

Through the shuffling of predefined modular zinc finger domains with predictable target site recognition in vitro, we have generated a large repertoire of artificial transcription factors with five zinc finger domains (TFZFs). Here we report an effective strategy for the selection of ATF libraries by coupling expression of transcriptional activators of the promoter of interest to the enhanced production of retroviral vector particles transferring the TFZF encoding gene. Using this strategy, we successfully selected specific TFZFs that upregulate the expression of the γ-globin promoter. Selected transcription factors induced the expression of γ-globin when coupled to an activation domain and reduced expression when linked to a repression domain. This new retroviral approach might be used to select other TFZFs but might also be generalized for the selection of other protein and small-molecule interactions.

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Figure 1: Schematic illustration of the selection strategy.
Figure 2: Selected TFZF libraries show increased GFP-positive transduced cells after each round of selection.
Figure 3: Selected TFZFs shows repression of γ-globin promoter expression when fused to the repressor domain KRAB.
Figure 4: Activation of human γ-globin in β-YAC bone marrow cells (BMCs) containing selected zinc fingers, as measured by RT-PCR.

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Acknowledgements

Thanks to F.C. Costa and R. Neades for technical assistance with the BMC transfections and expression assays. This study was supported by the Skaggs Institute for Chemical Biology and in part by US National Institutes of Health Grants RO1 DK61803 and R01GM065059 (C.F.B.) and R01 DK061804 (K.R.P.).

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U.T., K.R.P. and C.F.B. designed the research; U.T., K.R.P., B.G. and H.F. performed the experiments; U.T., K.R.P. and C.F.B., wrote the manuscript, which all authors commented on.

Corresponding author

Correspondence to Carlos F Barbas III.

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Supplementary Table 1 and Supplementary Methods (PDF 408 kb)

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Tschulena, U., Peterson, K., Gonzalez, B. et al. Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production. Nat Struct Mol Biol 16, 1195–1199 (2009). https://doi.org/10.1038/nsmb.1677

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